J. Sci. Fd Agric. 1978, 29, 1030-1036

A Study of the Nutritive Value of Toprina G in the Diet of Two Hybrid Strains of Caged Laying Hens

Norman Jacksonab and Griffith McC. Kirkpatrickb

aAgricuItura1 and Food Chemistry Department, The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, and Department of Agriculture for Northern Ireland

(Manuscript received 10 May 1978)

An experiment is reported in which the hydrocarbon-grown yeast Toprina G was used at levels of 0, 20, 40, 60, 80, 100 and 120 g kg-l of diet to replace mainly soy- bean meal in a fishmeal-free control diet fed to two lightweight hybrid strains of caged layers. The Toprina G had a very low Se content (14 pg kg-l) and a high Mn content (325 mg kg-l). Replacement of the soy-bean meal of the control diet and also, at the highest level of Toprina G addition, the meat and bone meal by the hydrocarbon-grown yeast did not statistically affect total egg weight or mean egg weight significantly (total egg weight/total number of eggs laid). Total food intake tended to be higher for the birds fed the diets containing Toprina G although for the full experimental period this effect was not statistically significant. For one of the hybrid strains the food conversion efficiency was poorer for all the treatments receiving Toprina G than for the control. This effect was statistically significant at the 60 and 100 g Toprina G inclusion per kg of diet. The results indicate that Toprina G may be used as the major protein source in the diet of caged layers up to a level of 12 7:.

1. Introduction

Toprina G is a dried yeast material of high crude protein (CP) content and adequate metabolisable energy (ME) content obtained by the degradation of n-paraffins (C11LC18) produced in the oil industry. Toxicological investigations have shown it to be free from undesirable side effects.1-4

Although the CP content of Toprina G is high its amino acid content is nutritionally defective in that it has a relatively low content of sulphur-containing amino acids. This is compensated for to some extent by the high availability of the methionine as shown by microbiological assay.5 At the present time the deficiency of sulphur-containing amino acids is not a major problem since synthetic DL-methionine is currently available at a price which makes its use as a dietary supplement economic. The amino acids present in Toprina G have been shown to be highly digestible.6 The lysine present in Toprina G has been shown to have a high availability7 as was previously indicated by chemical assay.s

Various experiments have been reported to show that yeasts grown on hydrocarbons can be used as a complete or partial replacement for the more conventional protein sources in diets for both pigs and poultry. The results of much of this work have been summarised by Shacklady.9 Other workers10 have shown that Toprina G, fed to broiler chickens from 1 day old to 8 weeks of age, results in satisfactory liveweight gains and feed conversion when present at levels of 50 and 100 g kg-1 of diet. Shannon et al.ll found that Toprina G fed to layers at up to 100 g kg-1 of diet resulted in lower egg production and lower feed intake by medium hybrids while the food conversion efficiency of both light and medium hybrids was unaffected. Yoshida et ~ 1 . ~ 2 have shown that yeasts produced on n-paraffins can maintain egg production and egg weight, and have no adverse effect on feed intake when fed up to 150 g kg-l in the diet of laying hens. However, in other Japanese

0022-5142/78/1200-l030 $02.00 0 1978 Society of Chemical Industry

1030

Nutritive value of Toprina G to hens 1031

experiments egg weight was decreased as a result of the yeast inclusion in the diet.13 It has been reported14 that the productivity characteristics of laying hens were not affected when 10 :< Toprina G was used to replace the soy-bean meal protein in layers' diet which contained no animal protein.

It was found that when the fishmeal and soy-bean meal in a layers' diet were replaced by levels of up to 17.6% Toprina G, the latter treatments resulted in lower percentage egg production, total egg weights, mean egg weights and higher mortality than a control treatment.15

The experiment reported below was designed to compare, for two hybrid strains of caged layer, the efficiency, for egg production, of a basal diet containing no fishmeal but having soy-bean meal as the main protein source and with a 4 % inclusion of meat and bone meal with a series of diets containing increasing amounts of Toprina G as a protein source.

2. Experimental

The laying experiment commenced in August 1975 and finished in June 1976, and was divided into eleven 28-day periods.

Two hundred and eighty-eight birds, comprising equal numbers of two white, lightweight strains of hybrid pullets (H and N Nick Chick-referred to as Hybrid A; Babcock B300-Hybrid B) were housed in single-hen cages with individual feed troughs and nipple drinkers and maintained on a lighting programme of 17 h of light and 7 h of darkness. The battery house was unheated, the maxi- mum recorded summer temperature being 28 "C and the minimum recorded winter temperature being 4°C. The birds were fed a commercial chick mash from 1 day old, changed to a growers mash at 7 weeks of age and to a commercial layers' mash at 18 weeks of age. They were caged at 18 weeks of age and fed the experimental diets ad libitum from 27 weeks of age. Recording commenced at this time. Daily egg production was recorded and the eggs were weighed twice weekly. The hens were weighed initially and at the end of each of the 28-day periods. The pullets of each hybrid strain were randomly divided into seven treatment groups and each group fed one of the experi- mental diets. The diets were a control diet in which the principal protein concentrate was extracted soy-bean meal and six other diets in which mainly the soy-bean meal was replaced by 20, 40, 60, 80, 100 and 120 g of Toprina G kg-1 of diet. The meat and bone meal was reduced from 40 g kg-1 in diets 1-6 down to 10 g kg-l in diet 7. The diets were pelleted to minimise any possible effect on intake of the density differences in the unpelleted material which are seen in Table 2.

The diets were designed to be isonitrogenous and isocaloric with respect to ME. The calculated CP content of all diets was 159 g kg-l, the calculated ME values 11.74 MJ kg-l and the calculated contents of Ca and P were 31 and 6.1-6.4 g kg--l, respectively. The calculated lysine contents were 7.3-8.0 g kg-1, the calculated methionine contents 2.7 g kg-l and the calculated methionine plus cystine contents 5.2-5.4 g kg-l.

Mean analytical values for the Toprina G , soy-bean meal and meat and bone meal used are presented in Table 1. The amino acid contents of these high-protein ingredients and of the seven diets were determined on a Technicon Autoanalyser subsequent to open-flask acid hydrolysis and the values obtained left uncorrected for any losses during the hydrolysis.

The composition, determined ME and determined dry matter, CP, crude fibre (CF), Ca, P, Se and Mn contents of the experimental diets are given in Table 2 together with the density of the diet prior to pelleting. The ME content was determined by the total collection method using both types of hybrid. Se was determined by the method of Hall and Gupta16 and Mn by atomic absorption spectrophotometry subsequent to dry-ashing and solution in HCI/HN03.

The amino acid contents of the diets are presented in Table 3.

3. Results

The analyses of the Toprina G agree with previously published r e ~ u l t s . ~ The extremely low Se content of the Toprina G substantiated the value of 10 pg kg-1 cited eIsewhere.17 The Mn content of the Toprina G was extremely high compared with the soy-bean meal and the meat and bone meal.

1032 N. Jackson and G. McC. Kirkpatrick

Table 1. Mean determined analyses of Toprina G, extracted soy-bean meal and meat and bone meal used in the experimental diets

Soy-bean meal Meatand

Toprina G (extracted) bone meal

CP (g kg-9 Ether extract (g kg-1) Lipid material

(after acid hydrolysis) Gross energy (MJ kg-1) Ca (g kg-9 P (g kg-9 Se OLg kg-9 Mn (mg kg-1) Dry matter (g kg-1) Amino acidsa (g kg-l)

Alanine Arginine Aspartic acid Cystine Glutamic acid GI ycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

580.1 452.3 24.0 14.5

107.4 -

20.63 17.63 0.08 2.7

14.3 5.9 14 170

325 26 943 877

38.2 21 .o 31.9 40.3 62.5 59.4

8.9 9.6 82.6 84.4 32.3 21 .o 12.7 13.5 29.5 23.7 46.0 40.7 47.5 33.3 6.6 4 .1

28.7 27.3 27.9 30.5 28.7 24.7 29.3 18.3 21.9 18.4 34.0 24.3

461.6 139.1 -

16.82 102.8 35.6

210 34

927

31.1 34.6 37.9

1 . 3 56.3 60.1 6.9

15.5 32.3 22.2 4.6

17.5 51.9 24.6 16.6 10.1 25.9

“Uncorrected for losses during acid hydrolysis.

There were no treatment or breed differences in mortality, the average mortality over the laying period being 8 %.

The results for egg production, egg weight, food intake and efficiency of food conversion of the survivors, expressed as bird means, are presented in Table 4.

Total food intake was not significantly affected by treatment although in most cases the mean total food intake by the birds receiving Toprina G in the diet was higber than for the birds offered the control diet. This effect on food intake attained statistical significance in periods 2 (P< 0.001) and 4 (P< 0.01).

The numbers of eggs produced were not statistically significantly affected by treatment. In the case of the hybrid A hens the mean total number of eggs was lower for all the Toprina G treatments than for the control group except for treatment 5 while for the hybrid B hens egg production was lower for the control than for all the Toprina groups except for treatment 7. The treatment 6 hybrid A hens layed markedly fewer eggs than did the hens of either breed on any other treatment, being 14.6 % poorer than the control group.

Total egg weight was not significantly affected by treatment. In all periods the mean total egg weight from the hybrid A hens fed Toprina G in the diet was lower than for those on the control diet, the effect being particularly marked in treatment 6 where there was a depression of 19% below the control value. For the hybrid B hens the mean total egg weight for all the treatment groups with added Toprina was higher than for the control group, except in the case of treatment 7 where it was 4.3 % lower.

Nutritive value of Toprina G to hens 1033

Table 2. Composition and determined analyses of the basal diet and the diets containing Toprina G

Diet

1 2 3 4 5 6 7

Constituents Maize meal 527.7 Ground wheat 160.0 Soy-bean meal 170.0 Meat and bone meal 40.0 Toprina G 0.0 Dried grass 20.0 Ground limestone 70.0 Dicalcium phosphate 5.0 Common salt 4.0 Vitamin-nlineral mixn 2.5 DL-methionine 0.8

Total 1000

Determined analyses Standard M E (MJ kg-1) 11.98 CP (g kg-9 161.7 Energy :protein ratio 74

[ME (kJ kg-l): CP (g kg-I)] C F (g kg-9 22.3 Ca (g kg-l) 31.1 P (g kg-9 5.2 Mn (mg kg-l) 88 Se (pg kg-9 69 Density before pelleting (g ml-1) Dry matter (g kg-l) 887

0.718

457.7 230.0 140.0 40.0 20.0 30.0 70.0 5.0 4.0 2.5 0.8

1000

11.97 160.6 75

22.6 35.3

5.3 89 59

89 1 0.713

455.0 242.7 110.0 40.0 40.0 30.0 70.0 5.0 4.0 2.5 0.8

1000

11.79 160.0 74

21.4 33.4

5.5 87 54

899 0.721

337.7 375.0 75.0 40.0 60.0 30.0 10.0

5.0 4.0 2.5 0.8

1000

11.90 162.7 73

20.9 33.2

5 .8 97 49

894 0.137

105.7 640.0 22.0 40.0 80.0 30.0 70.0 5.0 4 .0 2.5 0.8

1000

11.87 161.7 73

19.9 34.3

6.1 105 36

894 0.728

62.7 685.0

0.0 40.0

100.0 30.0 70.0

5.0 4.0 2.5 0.8

1000

11.94 162.7 73

21.4 34.1 6.1

110 34

895 0.733

41.7 700.0

0.0 10.0

120.0 30.0 75.0 10.0 4 .0 2.5 0.8

1000

11.84 159.2 74

21.2 29.2 5.4

123 25

890 0.741

OThe vitamin-mineral supplement supplied (kg-l diet) 1 .7 mg retinol; 35 pg cholecalciferol; 4.0 mg or-tocopherol; 2 mg menadione' sodium bisulphite; 2.5 mg riboflavin; 4.0 pg vitamin B12; 10.0 mg nicotinic acid; 5.0 mg pantothenic acid; 100 mg choline chloride; 16 mg Fe; 3 mg Co; 7 mg Cu; 60 mg Zn and 2.5 mg I.

Table 3. Amino acid composition of the basal diet and the Toprina G-containing diets

Diet

1 2 3 4 5 6 7

Toprina G in diet (g kg-1) Amino acidsa (g kg diet-1)

Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

Dry matter (g kg diet-1)

0 20 40 60 80

8.7 8.4 8.6 9 .6 9.9 9.6

15.7 15.0 14.3 2.8 2.3 2.5

21.9 26.3 27.2 8.3 7.8 8.5 4 .1 3.9 3.8 7.0 6.5 6.6

14.3 13.3 13.4 8.3 8.0 1 .9 2.5 2.1 2.4 7.1 7.2 7.3

13.1 12.1 12.8 7.4 7.0 7.1 6.2 6.0 6.0 5.8 5.1 5.4 8.1 7.3 7.7

887 89 1 89 1

8.8 9.6

13.7 2.5

28.9 8.6 3.9 6.7

13.2 8.2 2 .4 7.4

13.4 7.3 6.0 5.6 8.0

894

8.0 9.2

12.4 2.5

31.2 8.5 3.7 6.5

11.7 7.9 2 .4 1 .3

13.5 7.1 5.8 5.3 7.8

894

100

8 .8 9.1

12.9 2.1

30.5 9.0 3.7 6.8

11.9 8.5 2.4 7.3

12.7 7.2 5.9 5.1 8.3

895

120

7 .9 8.3

12.3 2.8

28.2 7.6 3.3 6.4

11.1 8.2 2.1 7.3

12.1 7.0 5.9 5.2 8.0

890

a Uncorrected for losses during acid hydrolysis.

1034 N. Jackson and G. McC. Kukpatrick

Table 4. Mean egg production, egg weight, fcod intake and food conversion results for control and Toprina G-fed hens

Diet aApproximate s.e. of a mean

Breed 1 2 3 4 5 6 7 (251 df)

Toprina G i n diet (g kg;-l) No. of observations

Mean eggs per bird

Mean egg production I: ;<)

Mean total egg weight (kg)

Mean period mean egg weight (g)

Total egg weight fg) Eaas oer bird

Totalffoid intake (kg)

Daily food intake (g)

Food conversion (kg eggs kg-1 food)

A B A B A B A B A B A B A B A B A B -

0 20 40 60 80 100 120 19 18 19 17 16 18 19 19 20 20 21 19 20 20

225 221 221 219 229 192 222 224 249 229 235 243 239 219

73.0 71.8 71.9 71.3 74.3 62.3 72.2 72.8 80.7 74.3 76.3 78.8 77.7 71.2 13.94 13.75 13.66 13.05 13.47 11.33 13.91 13.58 15.02 13.81 13.78 14.77 14.15 13.00 62.4 62.6 62.1 59.5 59.5 59.4 62.9 61.3 60.7 60.3 59.0 61.2 59.4 59.6 62.0 62.1 61.6 58.9 59.1 58.3 62.5 60.9 60.6 59.9 58.5 61.0 59.0 59.0 34.29 34.68 35.13 36.16 36.12 35.32 36.07 34.89 35.52 35.89 34.85 36.30 37.24 36.40

111.3 112.6 114.0 117.4 117.3 114.7 117.1 113.3 115.3 116.5 113.2 117.9 120.9 118.2

0.406 0.393 0.387 0.353 0.373 0.317 0.385 0.390 0.424 0.382 0.395 0.408 0.380 0.357

- -

11.07

3.59

0.685

0.88

0.88

0.881

2.860

0.018

W e . correct for a mean of 19 birds. S.e. refers to A and B.

Mean egg weight (total egg weight/total number of eggs laid) was not statistically significantly affected by treatment although when analysed as the mean period mean egg weight (total weight of eggs per period/number of eggs per period) it was affected by treatment (P< 0.01). The mean was lower for most of the treatments receiving the Toprina G and the effect attained statistical signifi- cance (P< 0.05) in the case of the hybrid A hens fed diets 4, 5 and 6. The mean period mean egg weight was lower ( P < 0.05) for the B hybrids than for the A hybrids.

Food conversion efficiency was significantly affected by treatment (P< 0.05) being, in the case of the hybrid A hens, poorer for all the treatments receiving Toprina G, this effect attaining signi- ficance for treatment 4 (P<0.05) and treatment 6 (P<O.OI). None of the treatments caused a statistically significant effect on food conversion efficiency in the case of the hybrid B hens.

The mean initial and final bodyweights, ME intakes, ME conversion results, expressed as kg eggs per MJ of ME intake, and CP intakes are presented in Table 5 together with the average body- weights over the period of the experiment. This latter figure was obtained for each treatment from a regression equation for the bodyweight attained at 28-day intervals over the experimental period.

4. Discussion and conclusions

The experiment was initiated to obtain further information on the nutritive value of Toprina G, both by chemical analyses and by observing the effect of its inclusion in the diet of two hybrid strains of caged layer. The two hybrid strains were selected in the anticipation that the differential in the final bodyweights of the hybrid A and hybrid B hens would be greater than proved to be the case. This lack of appreciable bodyweight difference is considered to be a deficiency in the experi- mental design.

The decision to pellet the diets was prudent in the light of results subsequently presented by van Weerden et al.17 who found for both semisynthetic and practical type diets fed to broilers that when the diets were pellefed no distinct weight differences between the Toprina G and fish meal control

Nutritive value of Toprina G to hens 1035

Table 5. Mean bodyweight, bodyweight gains, ME intake and ME utilisation data

Diet aApproximate s.e. of a mean

Breed 1 2 3 4 5 6 7 (251 df)

Toprina Gin diet (g kg-1) Bodyweight (survivors)

Initial (kg) A

Final (kg) A

of survivors (9) B Average bodyweight A

(survivors) over the B

B

B Mean bodyweight increase A

experiment period (kg) Mean daily ME intake (MJ) A

B Mean ME conversion A

(kg eggs MJ -l of ME) B Mean daily CP intake (g) A

B

0 20 40 60 80 100 120

1.45 1.54 1.77 1.92

249 338

I .66 1.80

1.60 1.45 1 . 8 8 1.80

226 289

1.81 1.71

1.52 1.46 1.74 1.83

177 340

1.71 1.71

1.53 1.47 1.85 1.80

238 283

1.74 1.70

1.51 1.48 1 . 8 1 1.87

263 320

I .72 1.74

1.47 1.47 1.71 1.85

230 352

1.65 1.74

1.49 0.039 1.46 1.73 0.053 I .85

179 324

1.67 0.043 1.72

1 . 3 3 1 . 3 5 1 . 3 5 1.40 1.39 1.37 1.39 0.034 1.36 1.38 1 . 3 8 1.34 1.40 1.44 1.40 0.034 0.033 0.033 0.030 0.031 0.027 0.033 0.0015 0.033 0.035 0.032 0.033 0.034 0.032 0,030

18.0 18.1 18.2 19.1 19.0 18.7 18.6 18.3 18.5 18.6 18.4 19.1 19.7 18.8

- ~ ~~~

S.e. correct for a mean of 19 birds. S.e. referes to A and B.

group were detected although Toprina G caused a distinctly poorer weight gain when the diets were fed as a meal.

The amino acid analysis of the Toprina G shows the low content of sulphur containing amino acids and the relatively high lysine content which is characteristic of this materiaLg Even with the addition of a DL-methionine supplement to the diets, the methionine plus cystine contents were scarcely more than adequate for the high-producing laying hen.18 However, the true methionine and cystine contents were probably considerably higher than the analyses indicate since destruction of these does occur under the conditions of hydrolysis used. In addition, the requirement of the hens should have been lower than the maximum since the hens were past peak production when the experiment started. The high lysine content of the Toprina G permitted the formulation of all the diets, even at the highest level of Toprina G inclusion, without any L-lysine supplementation. All the other amino acids were present at levels which are accepted as being adequate for the laying hen.18

The determined ME, CP, Ca and P contents of the diets were acceptably close to the calculated values. The ME values indicate agreement with the value of approximately 12.7 MJ kg-1 for the classical ME of Toprina G.6 The Se content of the Toprina G was found to be quite low so that as Toprina G replaced the soy-bean meal and meat and bone meal, the Se content of the diet fell from 69 to 25 pg kg-1. The low Se content of Toprina G has been noted previouslyl7 where it was reported that the addition of 0.2 mg kg-1 Se to practical type diets containing Toprina G resulted in a sig- nificant improvement in the growth of broilers. In the present experiment there is no indication that the decrease in Se content has had any adverse effect on production. Another interesting feature of the Toprina G is the high manganese content and in diet 7 replacement of the soy-bean meal and three-quarters of the meat and bone meal by Toprina G resulted in a 40% increase in dietary man- ganese content. The value of 325 mg Mn kg-1 for the Toprina G is considerably higher than that of 250 mg kg-1 reported for an unspecified paraffin-grown y e a ~ t . 1 ~

The presence of Toprina G in the diet was, except in one case (hybrid B birds, diet 4), associated with an increased feed intake (average 3.5 %) and this was clearly reflected in the ME intake (average

1036 N. Jackson and G. McC. Kirkpatrick

increase 2.8 %). I1 seems probable that this effect, which was not statistically significant, is never- theless, a real effect. For the light hybrids the combined effect of a slight fall in total egg weight together with the slight increase in feed intake when Toprina G was included in the diets resulted in a poorer mean feed conversion, an effect which would appear to be real although it only attained significance for two treatments. There appears to be no obvious reason for the poor egg production by the light hybrids in treatment 6. The indication of a slight reduction in mean egg weight, especially for the B hybrids., is in agreement with the observation of Seno et aZ.13

The results indicate that Toprina G can be used in a layers’ diet up to a level of 120 g kg-1 without any markedly adverse effect on egg production expressed either as number of eggs or total weight of eggs produced over the period of the laying trial. The Toprina G was replacing mainly soy-bean meal, and at the top level of inclusion was replacing all of the soy-bean meal contributing 43.7 % of the total CP of the diet. Although all of the diets had animal protein incorporated as meat and bone meal, the content of this was relatively low, and fell to 1 % of the diet, supplying only 2.9% of the dietary CP at the top level of Toprina G inclusion.

The results indicate that for both the hybrid strains used Toprina G could be supplied as the major protein source at an inclusion of up to 12%, although for the hybrid A birds there was an indication of a decrease in feed conversion efficiency. The laying trial results are in general agreement with those of other worker~.ll-~5

Acknowledgements Thanks are due to the Trustees of the Agricultural Research Institute, Hillsborough, for providing facilities for the laying trial, to colleagues in the Biometrics Division and to Mrs R. Park and Mr P. Shearer for technical assistance.

References 1. Llewelyn, D. A. B. Proc. Conf. on Microbiology Institute of Petroleum, London, 1968, p. 63. 2. Shacklady, C. A. 3rd Int. Congr. Food Sci. Tech. Washington, DC, 1970. 3. de Groot, A. P.; Til, H. P.; Feron, V. J . Fd Costnet. Toxicol. 1970. 8, 267. 4. de Groot, A. P.; Til, H. P.; Feron, V. J. Fd Costnet. Toxicol. 1970. 8, 499. 5. Barber, R. S.; Braude, R.; Mitchell, K. G.; Myers, A. W. Br. J . Nutr. 1971, 25, 285. 6. Shannon, D. W. F.; McNab, J. M. J . Sci. Fd Agric. 1973, 24, 27. 7. Lewis, D.; Boorman, K . N.; Morgan, D. J. Proc. 10th lnt. Congr. Aniin. Prod. Versailles, EAAP, 1971. 8. Roach, A. G.; Sanderson, P.; Williams, D. R. J . Sci. Fd Agric. 1967, 18, 274. 9. Shacklady, C. A. Wld Rev. Niitr. Diet. 1972, 14, 154.

10. Shannon, D. W. F. ; McNab, J. M. Br. Poult. Sci. 1972, 13, 267. 11. Shannon, D. W. F.; McNab, J. M.; Anderson, G. B. J. Sci. Fd Agric. 1976, 27, 471. 12. Yoshida, M.; Tada, M.; Bansho, H.; Matsuchima, M.; Koba, K.; Iino, M.; Umeda, 1. Jup. Poult. Sci. 1973.

10, 63. 13. Seno, F.; Tada, M.; Iwamoto, T.; Murata, T.; Kawasaki, A. Jup. Poult. Sci. 1973, 10, 189. 14. Castello, J. A. 5th Eur. Poult. Conf. Malta, 1976, p. 171. 15. Svetic, M.; Binicki, M.; Tezulati, Z.; Grle, A.; Lugar, I.; Pavlov, M.; Peraic, 1.; Dubravec, T.; Roseg, D.;

Hadsiosmanovic, M. Unpublished work. 16. Hall, R. J.; Gupta, P. L. Analyst, Lond. 1969, 94, 292. 17. van Weerden, E. J . ; Schutte, J. B.; Shacklady, C . A. 5th Eur. Poult. Conf. Malta, 1976, p. 181. 18. The Nutrient Requirements of Farm Livestock. 1. Poultry (ARC), HMSO, London, 1975. 19. Bolton, W.; Blair, R. Poultry Niltrition, Bull. No. 174 Ministry of Agriculture, Fisheries and Food, 1974,

HMSO. London.